Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by the degenerative loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Studies of cellular and animal models and postmortem PD patient brains reveal that synaptic dysfunction triggered by environmental and genetic stress is an early event in PD pathogenesis. However, the precise and key mechanisms that underlie synaptic dysfunction in PD remain to be defined. The vacuolar (H+)-ATPase (V-ATPase) is an ATP- dependent proton pump involved in acidifying synaptic vesicles and plays a critical role in multiple steps of synaptic vesicle life cycle such as fusion with pre-synaptic membrane and neurotransmitter release/reloading. Our preliminary studies show that synaptic V-ATPase may be controlled by chaperone-mediated autophagy (CMA), a lysosome-based process specialized in disposing oxidized or damaged proteins for degradation to maintain cellular function under stress. Notably, CMA is inhibited by multiple stress conditions associated with PD including aging, neurotoxic stress, oxidative stress, ER stress, and genetic stress. However, whether and how cellular stress signals and CMA engage V-ATPase to modulate synaptic function is still unknown. In the current project, by utilizing multiple model systems, including cutting-edge human induced pluripotent stem cell (iPSC) model, in vivo rodent models (ER and genetic stress of rodent brains), and postmortem PD patient brains, we aim to determine whether CMA directly degrades damaged components of synaptic V-ATPase to maintain synaptic vesicle function under stress, and whether loss of adequate CMA activity impairs synaptic function in PD. First, we will determine biochemically, cell biologically, and electro-physiologically if CMA directly regulates synaptic V-ATPase and synaptic function in DA neurons derived from human iPSCs. Second, we will test if multiple stress conditions related to PD regulate synaptic V-ATPase and function via CMA in human iPSC-derived DA neurons. Last, we will assess the regulation and role of CMA?V-ATPase?synaptic vesicle pathway in multiple PD animal models and in postmortem brains from PD patients. Our proposed study will significantly advance our understanding of how stress regulates synaptic vesicle, reveal a key pathogenic mechanism underlying synaptic failure in PD, and offer new opportunities for developing diagnostics/biomarkers and more effective prevention and treatment strategies for the disease.